biomolecules Article Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor Xiaoyuan Ma , Eléonore W. E. Verweij, Marco Siderius, Rob Leurs and Henry F. Vischer * Division of Medicinal Chemistry, Faculty of Science, Amsterdam Institute of Molecular and Life Sciences, Vrije Universiteit Amsterdam, 1081 HZ Amsterdam, The Netherlands; [email protected] (X.M.); [email protected] (E.W.E.V.); [email protected] (M.S.); [email protected] (R.L.) * Correspondence: [email protected] Abstract: The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits β-arrestins upon phosphory- lation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling. Keywords: histamine; H R; GPCR; GPCR-interacting proteins; membrane yeast two hybrid; TSPAN4; Citation: Ma, X.; Verweij, E.W.E.; 4 Siderius, M.; Leurs, R.; Vischer, H.F. tetraspanin Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor. Biomolecules 2021, 11, 1127. https:// doi.org/10.3390/biom11081127 1. Introduction Histamine is a key mediator of allergic inflammation and is released from mast cells Academic Editors: Emanuela Masini and basophils upon allergen binding. The histamine H4 receptor (H4R) is predominantly and Laura Lucarini expressed on immune cells and mediates histamine-induced chemotaxis and production of inflammatory cytokines [1,2]. Importantly, H4R-deficient mice revealed a role of this Received: 14 July 2021 receptor in pruritus, dermatitis, asthma, and arthritis disease models [2]. Consequently, Accepted: 28 July 2021 the H4R has been recognized as potential anti-inflammatory drug target, and selective Published: 30 July 2021 antagonists are currently in clinical trials to counteract atopic dermatitis, psoriasis, allergic rhinitis, bronchial allergen challenge, asthma, and rheumatoid arthritis [2]. Publisher’s Note: MDPI stays neutral The H4R is a G protein-coupled receptor (GPCR) that activates heterotrimeric Gαi with regard to jurisdictional claims in protein-mediated intracellular signal transduction resulting in decreased cAMP produc- published maps and institutional affil- tion [3–7], increased Ca2+ mobilization [8–10], activation of extracellular-signal regulated iations. kinase (ERK)1/2 [11–15], as well as cytoskeletal changes [8]. Similar to most other GPCRs, the H4R can interact with G protein receptor kinases (GRKs) and β-arrestins that are in- volved in receptor internalization and consequently the regulation of agonist-induced G protein signaling [14,15]. Copyright: © 2021 by the authors. GPCRs have been found to also interact with other membrane and/or intracellular Licensee MDPI, Basel, Switzerland. proteins in addition to heterotrimeric G proteins, GRKs, and β-arrestins. These so-called This article is an open access article GPCR interacting proteins (GIPs) can mediate and/or modulate GPCR signaling and/or distributed under the terms and trafficking [16–18], and have been identified by various biochemical (e.g., pull-downs from conditions of the Creative Commons cell lysates using Glutathione S-transferase (GST) or His6-tagged GPCR C-tails [19,20], or Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ HDL-reconstituted GPCR [21]), cell biological (e.g., ascorbate peroxidase (APEX)-catalyzed 4.0/). proximity labeling in living cells followed by affinity chromatography, enzyme digestion, Biomolecules 2021, 11, 1127. https://doi.org/10.3390/biom11081127 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1127 2 of 16 and mass spectrometric analysis [22–24]), and genetic approaches (e.g., membrane yeast two-hybrid (MYTH) [25–31]). In this study, the MYTH approach was used to identify GIPs of the human H4R from an unstimulated Jurkat T cell cDNA library [32]. In short, the H4R was fused at the N-terminus to the yeast STE2 leader sequence to target the fusion protein to the membrane of yeast, whereas the C-terminal half of ubiquitin (Cub) and the LexA-VP16 transcriptional activator were fused in frame to the intracellular C-terminal tail of H4R. The prey proteins encoded by the naïve Jurkat T cell cDNA library were N-terminally tagged with the N-terminal half of ubiquitin that contains an isoleucine 13 to glycine mutation (NubG) to prevent spontaneous ubiquitin reconstitution (Dualsystems Biotech, Switserland) (Figure1A). Interaction of unliganded H 4R-Cub-LexA-VP16 (bait) with NubG- tagged prey proteins in yeast allows reconstitution of a pseudoubiquitin moiety, which is subsequently recognized by endogenous ubiquitin-specific proteases resulting in the cleavage of the LexA-VP16 transcriptional regulator. The released transcriptional regulator translocates into the nucleus and activates transcription of LexA-promoter fused reporter genes for selection (LacZ, HIS3, ADE2) (Figure1B). Figure 1. Schematic overview of the split-ubiquitin membrane-based yeast two-hybrid (MYTH) assay. (A) Diploid yeast co-expressing the membrane targeted H4R-Cub-LexA-VP16 bait and cytosolic (depicted) or membrane-associated N- terminally tagged NubG prey proteins was generated by mating of haploid yeasts expressing the individual bait and prey constructs. (B) Interaction of the H4R-Cub-LexA-VP16 ‘bait’ with NubG-protein ‘prey’ results in functional reconstitution of a pseudo-ubiquitin, which is subsequently recognized by cytosolic ubiquitin-specific proteases leading to the cleavage of the LexA-VP16 transcriptional activator and the expression of (HIS3, ADE2, LacZ) reporter genes. (C) Subcellular localization and biological function summary of the 43 hits were retrieved from the Uniprot database (https://www.uniprot.org; accessed on 24 June 2021). Gene names are indicated and full description of proteins with Uniprot codes are presented in Supplementary Table S1. Hits in bold have been found to interact with other GPCRs in MYTH screens (see Supplementary Table S1), whereas frequent MYTH screen hits are indicated in italics. ER = endoplasmic reticulum; PM = post-translational modification (D) STRING analysis (v.11.0; https://string-db.org; accessed on 25 June 2021) of hits from the MYTH screen of H4R (bait) on an unstimulated Jurkat T cell DUALmembrane cDNA library. Known interactions between proteins in the STRING database are indicated by connecting lines. The thickness of the line represents the degree of confidence prediction of the interaction. Biomolecules 2021, 11, 1127 3 of 16 This MYTH approach identified 43 novel GIPs for the H4R. The interaction of one potential GIP (i.e., human tetraspanin 4; TSPAN4) was further validated in transfected HEK293T cells using bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation. In addition, the conse- quence of this H4R-TSPAN4 interaction was evaluated in ligand binding and H4R-mediated G protein activation assays. 2. Materials and Methods 2.1. Materials and Reagents Fetal bovine serum was obtained from Bodinco (Alkmaar, The Netherlands), and penicillin/streptomycin was purchased from GE Healthcare (Uppsala, Sweden). Dul- becco’s Modified Eagle’s Medium (DMEM, #41966-029), Dulbecco’s phosphate-buffered saline (DPBS, #D8662), Hanks’ Balanced Salt Solution (HBSS, #14025-050), trypsin-EDTA, PierceTM bicinchoninic acid (BCA) protein assay kit, GeneJET gel extraction kit, and GenJET plasmid Miniprep kit were purchased from Thermo Fisher Scientific (Waltham, MA, USA). [3H]-histamine (specific activity 20.0 Ci/mmol), Microscint-O scintillation liquid, GF/C filter plates were purchased from PerkinElmer (Groningen, The Netherlands). NanoGlo® was bought from Promega (Madison, WI, USA). 40,6-diamidino-2-phenylindole (DAPI), Whatman® Westran® PVDF membranes, cOmplete™, EDTA-free Protease Inhibitor Cock- tail and histamine·2HCl were bought from Sigma-Aldrich (St. Louis, MO, USA). Linear polyethylenimine (PEI, 25-kDa) was obtained from Polysciences (Warrington, FL, USA). Monoclonal Anti-HA (rat) antibody (#11867423001) was obtained from Roche (Roche Di- agnostics; Mannheim, Germany). Goat polyclonal antibody to Venus (#orb334993) was bought from Biorbyt Ltd. (Cambridge, UK). Goat anti-Rat IgG (H+L) Secondary Antibody and Rabbit anti-Goat IgG (H+L) Secondary Antibody were from Bio-Rad Laboratories (Hercules, CA, USA). All other reagents were of analytical grade and obtained from con- ventional commercial sources. 2.2. MYTH Constructs and Screen The MYTH bait vector pBT3-STE-H4R was generated by PCR. A SfiI fragment con- taining the full open reading frame of H4R was subcloned